Interferons (referred to as IFN hereinafter) so far known can be classified into three large groups, i.e. IFN-.alpha., IFN-.beta. and IFN-.gamma.. IFN-.alpha. is mainly produced from leukocytes, IFN-.beta. from fibroblasts and IFN-.gamma. from T-lymphocytes. These IFNs have been noted as biologically active substances having anti-virus activity, activating activities upon natural killer cells and macrophages, anti-tumor activity, and the like. However, the conventional methods for obtaining IFNs by isolation from leukocytes and cultured cells cannot provide them sufficiently.
Recombinant DNA technology has now been developed to the extent that the mass production of substances which are secreted only in a small amount in cells of higher animals and are hard to isolate such as IFN has become possible by using microorganisms. For example, mRNAs of IFN-.beta. and IFN-.alpha. were respectively isolated from cells and DNA complementary to the mRNA (cDNA) was synthesized enzymatically and cloned in Escherichia coli [Taniguchi, et al.: Proc. Jap. Acad., 55 (B), 464-469 (1979), Nagata, et al.: Nature 284, 316-320 (1980)].
As for IFN-.gamma., there has been a report that it has a stronger cell-inhibiting activity than other IFNs based on the experiment using animal cells [B. Y. Rubin and S. L. Gupta: Proc. Natl. Acad. Sci., USA, 77, 5928-5932 (1980)]. Furthermore, cloning of an IFN-.gamma. cDNA into Escherichia coli and determination of its base sequence were recently reported [P. W. Gray, et al.: Nature 295, 503 (1982), R. Devos, et al.: Nucleic Acids Research 10, 2487 (1982)].
The present inventors have independently cloned a DNA coding for IFN-.gamma. to obtain a clone coding for a novel IFN-.gamma. wherein, as apparent from the base sequence illustrated in Table 1, the ninth amino acid of the mature IFN-.gamma. reported by Devos, et al., Lysine (Lys) (AAA), is replaced with glutamine (Gln) (CAA). Further, the IFN-.gamma. gene was incorporated into vector pKYP-10 having tryptophan prompter (Japanese Published Unexamined Patent Application No. (110600/83) and mass production of the IFN-.gamma. in Escherichia coli has been achieved.
Thereafter, the present inventors have studied the production of derivatives of IFN-.gamma. polypeptide using the IFN-.gamma. gene illustrated in Table 1 as a starting material.
It was reported that deletion of 11 amino acids from the C-terminal of IFN-.alpha. decreased specific activity to one-third [A. E. Franke, et al.: DNA 1, 223-230 (1982)], whereas addition of 18 amino acids to the N-terminal of IFN-.alpha. did not change specific activity [R. M. King, et al.: J. Gen. Virol. 64, 1815-1818 (1983)].
Derivatives of IFN-.gamma. have not yet been reported. The present inventors have constructed a derivative wherein the third amino acid of IFN-.gamma. illustrated in Table 1, cysteine (Cys) was replaced with tyrosine (Tyr) (referred to as 3-Tyr-IFN-.gamma. hereinafter) and found that the specific activity was 2-4 times stronger than that of the parent IFN-.gamma.. Further, the derivatives wherein the Cys at the position 1 was replaced with serine (Ser) (1-Ser-IFN-.gamma.), the Cys at the position 3 was replaced with Ser (3-Ser-IFN-.gamma.), the Cys at the positions 1 and 3 were replaced with Ser (1,3-Ser-IFN-.gamma.) and N-terminal amino acids of IFN-.gamma. illustrated in Table 1 were deleted were constructed. Equivalent or more interferon activity was detected for all the derivatives compared with the starting IFN-.gamma..